Evolutionary pressures placed upon lentiviruses, such as HIV, and their affected hosts have resulted in a molecular arms race which has shaped both host immunity and pathogen immune evasion strategies. Central to this race is the HIV protein Virion Infectivity Factor (Vif), an immunomodulatory protein critical to the replicatio cycle of nearly all lentiviruses. The primary function of Vif is to counteract the antiviral effect of the host APOBEC3 (A3) innate immune proteins. A3 proteins are restriction factors that inhibit lentiviral replication by inducing hypermutation of the viral genome. Vif antagonizes A3 by hijacking a cellular Cullin-RING ubiquitin ligase, resulting in the ubiquitination and subsequent targeting of APOBEC3 for proteasomal degradation. Despite the central role of Vif in the HIV replication cycle and an extensive body of literature describing HIV pathogenesis, we still do not have a clear molecular understanding of how Vifs bind and ubiquitinate A3. This research plan seeks to combine biochemistry, enzymology, and structural biology to understand how Vif inhibits A3 at a molecular level, and determine how A3 inhibition relates to the evolutionary events that allowed HIV to jump from monkeys to humans. Lentiviral Vifs have evolved to specifically counteract the effects of the A3 from their animal hosts. To determine the biochemical basis for species-specific A3 recognition I will use a panel of HIV and SIV Vif E3 ligases and their respective A3G proteins to carry out binding and in vitro ubiquitination assays and address the question: is A3G species-specificity conferred during the ground state, binding to Vif, or the transition state, the catalytic step, of the enzyme catalyzed ubiquitination reactio? To provide the molecular determinants that govern Vifs' ability to confer species-specific recognition of A3G, I will utilize low- and high-resolution structural techniques that allow me to visualize the A3G-Vif interface at atomic resolution. Mutational analyses, using in vitro ubiquitination assays and cell-base infectivity assays, will be used to link structure to phenotype and validate the biological relevance of the structural model. The successful completion of these studies will provide snapshots of Vif from different stages of lentivirial evolution, and our combined structural and functional approach will provide detailed insight into the Vif- A3 interaction. Together these studies will allow us to see the Vif-A3 interaction, characterize the evolutionary steps required for species-species transmission into human populations, and ultimately aid in the development of innovative therapeutics to counter viral infection.